Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 392-399

Minireview



Enzymatic transesterification for biodiesel production
Shweta Shah, Shweta Sharma and M N Gupta*
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110 016, India
Received 9 June 2003; revised 1 September 2003
Biodiesel consists monoalkyl esters of long chain fatty acids. It is produced from vegetable oils or fats either by chemi-
cal transesterification or by lipase-catalyzed transesterification with methanol or ethanol. Biodiesel is a green fuel and can be
used as a blend with diesel or alone. Either way, it does not require any modification in engine design or storage facilities.
The enzymatic process offers several advantages over the chemical routes. The handicap of increase in process cost because
of the cost of the enzyme can be overcome by using efficient production process for enzyme and using reusable derivatives
of enzymes, such as immobilized enzyme. Numerous strategies available in the area of non-aqueous enzymology can be ex-
ploited during the enzymatic alcoholysis for biodiesel production. Some of the technical challenges and their possible solu-
tions are also discussed.
Keywords: Biodiesel, transesterification reactions, lipases, oil-extraction, enzymes in organic solvents
Introduction
The need for clean energy source is necessary be-
cause of the carcinogenic particulate emissions from
diesel engines, which cause pollution and global
warming. The depleting reserves of petroleum-based
products have also made scientists look for renewable
sources of energy. The concept of biodiesel addresses
these twin issues. Biodiesel is an efficient, clean, non-
toxic and biodegradable energy alternative to petro-
leum fuels
1
. Even though diesel is part of its name,
there are no petroleum or other fossil fuels in bio-
diesel. Biodiesel means a monoalkyl ester that
2
:
is derived from domestically produced vegetable
oils, renewable lipids, rendered animal fats or any
combination of those ingredients; and
meets the requirements of ASTM PS121, the pro-
visional specification for biodiesel.
The ASTM standard
3
, Indian standard
4
and stan-
dard D1NV51606
5
have been specified in the litera-
ture.
In fact, the use of vegetable oils (e.g. from palm,
soybean, sunflower, peanut, olive etc.) as such as
alternative fuels for diesel engine dates back to almost
a century
6
. The problem with direct use of such oils
arise because of their higher viscosity and lower
ignition quality as compared to diesel. The problems
are more severe in the case of direct-injection engines
than in the less efficient engines having
precombustion engines. In the case of direct engines,
very dilute blends of oils in diesel can be used
6
. Early
approach to improve the usefulness of the vegetable
oils consisted of pyrolysis of the oils. The pyrolysates
had lower viscosities and higher cetane numbers than
the parent vegetable oils
7
. Later work, of course,
showed that the production of fatty acid methyl esters
from the vegetable oils is a far more satisfactory
approach. This conversion of the oil into the esters is
essentially a transesterification reaction (Scheme 1). It
is these fatty acid methyl esters or ethyl esters, which
are collectively called biodiesel
8
. These
transesterification reactions can be catalysed by acid
or alkali
8
. Lately, considerable work has been carried
out on production of biodiesel by using lipases as
catalysts. The present review looks at these efforts
after discussing the advantages associated with
replacing chemical catalysts with enzymes in the con-
text of biodiesel production.

Sourcing starting material
One of the two reactants in the transesterification is
methanol or ethanol. As ethanol can be obtained from
renewable sources, it is the preferred starting material.
The degradation of starch by chemical or enzymatic
method produces glucose; its fermentation by whole
cell catalysis produces ethyl alcohol. At both steps,
free as well as immobilized biocatalysts have been

*Author for correspondence
Tel.: 91-11-26591503; 91-11-26596568
Fax: 91-11-26581073
Email: mn_gupta@hotmail.com
SHAH et al.: ENZYMATIC TRANSESTERIFICATION FOR BIODIESEL PRODUCTION


393

Scheme 1Biodiesel production from vegetable oils/fats by
transesterification

used. The fat or oil can be obtained normally from
seed or kernel from variety of plant sources.
Unfortunately, there is a gap between the produc-
tion and demand in the case of edible oil. Thus, a rea-
sonable approach has been to search for oils, which
are not used for edible purpose. Jatropha, a tropical
plant has been extensively studied for production of
biodiesel
9
. Its oil contains some toxic substances,
which makes it unfit for human consumption. It may
also be added that some people feel that using Jatro-
pha oil for biodiesel production diverts it from its ex-
isting uses for soap making, lighting and motive
power
10
.
The oils from tobacco
11
, mango kernel
12
, palm
8,13
,
soybean
7,14
, sunflower
15,16
, safflower
17
, cottonseed
18
,
rapeseed
19,20
, Jatropha
21
and beef tallow
22
have been
used for biodiesel production. Its preparations from
waste or spent palm oil
23
and restaurant grease
24
are
also worth mentioning. Shimada et al.
25
have men-
tioned, In Japan, 400000 tons of waste edible oils are
discharged yearly. In this regard, several local gov-
ernments in Japan have started collecting used frying
oils from households and have converted them to bio-
diesel fuel for public transportation. It is an example,
which is worth emulating.
Extraction of oil/fat from plant sources
Generally, three main approaches for extraction of
oil/fat have been used: hydraulic pressing, expeller
pressing and solvent extraction
26
. While pressing and
expelling can be used for oilseeds containing more
than 35% oil, solvent extraction with hexane can be
employed irrespective of the oil content
27
. However,
the main problem with solvent extraction has been the
production of volatile organic compounds, which
harm the ecosystem. During the last several years,
environmental concerns have led scientists to focus on
development of methods, which do not utilize organic
solvents. Aqueous oil extraction (AOE) consists of
breaking the oil bodies mechanically in the presence
of water and collecting the floating oil from the sur-
face
27
. The yields of oil, understandably, are generally
quite poor, but not insignificant. This encouraged the
development of aqueous enzymatic oil extraction
(AEOE), wherein enzymes like cellulase, hemicellu-
lase and proteases are used to free oil bodies en-
meshed in complex chemical structures by hydrolyz-
ing the latter
28,29
. In our lab, AEOE has given high
yields of 86 and 78% in the case of peanut and rice
bran, respectively
28,29
.
Recently, we have also described a novel approach
of oil extraction by three-phase partitioning
30
. It was
found that a mixture of salt and organic solvent mixed
with homogenous plant material led to the formation
of three phases: lower aqueous/water phase, interfa-
cial precipitate containing mostly protein and upper
organic phase, which contained oil. The oil can be
recovered from the organic solvent by evaporation or
freezing the mixture
31
. The technique has given prom-
ising results with oil from soybean
30
.

Benefits of biodiesel
In the past decade, biodiesel has been gaining
worldwide popularity as an alternative energy source
because of its many benefits.

1. Environmental benefits
Biodiesel is the only alternative fuel in the US to
complete EPA (Energy Policy Act) tier I health ef-
fects testing under section 211(b) of the clean air act,
which provides the most thorough inventory of envi-
ronmental and health effects attributes that current
technology will allow
32
. Unlike other clean fuels,
such as compressed natural gas (CNG), biodiesel and
other biofuels are produced from renewable agricul-
tural crops that assimilate carbon dioxide from the
atmosphere to become plants and vegetable oil. Bio-
INDIAN J. BIOCHEM. BIOPHYS., VOL. 40, DECEMBER 2003


394
diesel reduces the emission of carbon monoxide,
ozone-forming hydrocarbons, hazardous diesel par-
ticulate, acid rain-causing sulphur dioxide, smoke and
soot. It also lowers impact on marine environment by
reducing toxicity
32
.
2. Biodiesel is the only alternative fuel that runs in
any conventional, unmodified diesel engine. Also, it
does not require any changes in storage facilities,
which exist for storing petroleum-based diesel
32
.
3. Biodiesel can be used alone or mixed in any ratio
with petroleum diesel fuel
33
. The most common blend
is a mix of 20% biodiesel with 80% petroleum diesel,
or B20. Biodiesel has positive performance attrib-
utes such as increased cetane number, high fuel lu-
bricity, and high oxygen content, which may makes it
a preferred blending stock with future ultra-clean die-
sel
34
.
4. Biodiesel is safe to handle and transport because it
is as biodegradable as sugar, 10 times less toxic than
table salt, and has a high flash point of about 300 F,
compared to petroleum diesel fuel, which has a flash
point of 125 F
34
.
In fact, biodiesel is a proven fuel with over 30 mil-
lion successful US road miles, and over 20 years of
use in Europe
32
. It is the only alternative fuel that can
actually extend engine life because of its superior lu-
bricating properties
34
.

5. Energy security benefits
Many countries, which have to import oil and other
petroleum products become dependent upon other
nations. Their economy, thus, is extremely vulnerable
to any adverse effect of changes in political climate.
Biodiesel, produced from domestic sources, generates
self-reliance in a crucial area.

Biodiesel production
Biodiesel is produced from any fat or oil through a
refinery process called transesterification (Scheme 1).
Presently, the industrial production of biodiesel fuel is
performed by alcoholysis of waste oil using alkaline
catalysts. A by-product, glycerol, thus contains the
alkali, and hence has to be treated as a waste material.
Acid-catalysed transesterfication is another route for
biodiesel production, which is more suitable for glyc-
erides that have relatively high free fatty acid content
and more water. Aksoy et al.
35
reported that it was
necessary to perform transesterification under an
acidic condition when the oil composition was a low-
grade material containing sulphur etc.
Problems associated with chemical transesterification
of vegetable oil
High reaction temperature
36
.
Soap formation due to the presence of free
fatty acids in oil in case of base-catalyzed
transesterification reduces the ester yields
37
.
A major economic disadvantage in chemical
alcoholysis is that the purification of glycerol
(produced as a secondary product) is very dif-
ficult
16
.
Homogeneous catalyst removed with glycerol
layer cannot be reused.
For diesel fuel, ethyl ester is preferred because
ethanol can be produced from biomass and is
less toxic, but conventional alcoholysis with
ethanol gives low yields
21
.
In view of the above disadvantages associated with
the chemical transesterification for biodiesel produc-
tion, the enzymatic alcoholysis of oil/fat is considered
desirable. Table 1 compares chemical and enzyme-
based methods for biodiesel production
36
.

Enzymatic alcoholysis
To overcome problems associated with chemical
catalysis for production of biodiesel, enzymatic proc-
esses using lipases have been developed. Alcoholysis
by lipases is considered one of the most effective re-
actions for the production of biodiesel. Lipases (E.C.
3.1.1.3) hydrolyze triglycerides and can be isolated
from microorganisms, plants, mammals etc.
The lipase-catalyzed reactions can be classified as
follows
38
:

(i) Hydrolysis
R
1
COOR
2
+ H
2
O R
1
COOH + R
2
OH
Table 1Enzymatic and non-enzymatic methods for biodiesel
fuel production
36


Conditions Alkali-catalyzed Lipase-catalyzed

Reaction temperature 60-70C 30-40C
Free fatty acids in raw
materials
Saponified prod-
ucts
Methyl esters
Yields of methyl esters Normal Higher
Recovery of glycerol Difficult Easy
Purification of methyl
esters
Repeated washing None
Production cost of cata-
lyst
Cheap Relatively
expensive

SHAH et al.: ENZYMATIC TRANSESTERIFICATION FOR BIODIESEL PRODUCTION


395
(ii) Synthesis: reactions under this category can be
further divided:

Esterification
R
1
COOH + R
2
OH R
1
COOR
2
+ H
2
O

Transesterification
Alcoholysis
R
1
COOR
2
+ R
3
OH R
1
COOR
3
+ R
2
OH

Acidolysis
R
1
COOR
2
+ R
3
COOH R
3
COOR
2
+ R
1
COOH

The summary of biodiesel production from various
sources is given in Table 2. Biodiesel production, thus
involves using enzymes in non-aqueous media.

Non-aqueous enzymology and transesterification of
oils and fat
It is now well-established that the enzymes can be
used under nearly anhydrous conditions of neat or-
ganic solvents as reaction media
39,40
. The enzymes
under these conditions do require a minimum amount
of water, which is less than monolayer amount of wa-
ter molecules around the enzyme molecular surface.
Enzymes, in fact, become highly thermostable and
can be used at high temperatures. Lipases, for exam-
ple have been found to be biologically active at 100C
in nearly anhydrous organic solvent. The major appli-
cation of non-aqueous enzymology is synthesis by
hydrolases. Thus, in the absence of water, thermody-
namic reversibility pushes the enzyme-catalyzed reac-
tion in the reverse gear. A major limitation in the use
of enzymes in these media is their low catalytic effi-
ciency. This has been improved by immobiliza-
tion
41,42
, chemical modification
43
and protein engi-
neering
44
. For improving the catalytic efficiency as
well, protein engineering has been used
44
. Recently,
some workers have described salt activation for im-
proving catalytic efficiency
45
. Ultrasonication
46
and
microwave-irradiation
47
have also been used in a lim-
ited way. The cost of the enzymes is, in fact, always a
major deterrent in adopting their use in any biotech-
nological process.
The two major approaches, which are viewed as
possible partial solutions to the dilemma are: (i) effi-
cient bioseparation strategies; and (ii) enzyme immo-
bilization.
Table 2Summary of biodiesel production by transesterification of plant oils/fats

Route Oil source Catalyst Alcohol Yield (%)

Chemical Sunflower
7
KOH Methanol 96
Jatropha curcas
21
KOH Methanol 92
Ethanol 88.4
Crude palm
11
H
2
SO
4
Methanol 78
Enzymatic

(Free Enzyme) Soybean
22
Candida rugosa Methanol 30
Palm kernel
10
Psuedomonas cepacia Methanol
Sunflower
7
Psuedomonas fluorescens Methanol
Ethanol 82
Rapeseed
16
Candida rugosa 2-ethyl-1-hexanol 97
Mowrah,mango Mucor mehei C
4
-C
18:1
86.8-99.2
Kernel, Sal
19

Recycled Psuedomonas cepacia/ Ethanol 85.4
Restaurant grease
20
Candida antarctica


Enzymatic

(Immobilized)
Soybean
13
Candida antarctica Methanol 97
Safflower
14
Psuedomonas fluorescens
Cottonseed
15
Candida antarctica Methanol 87.4

INDIAN J. BIOCHEM. BIOPHYS., VOL. 40, DECEMBER 2003


396
Efficient bioseparation strategies
The major component of the production cost of any
enzyme is generally the expenditure on the down-
stream processing operations
48,49
. With this realiza-
tion, many novel bioseparation techniques have
emerged as a more efficient and economical altera-
tions. In classical downstream processing, a non-
selective precipitation with salt, organic solvent or a
water-soluble polymer (like PEG) is generally used to
concentrate the enzyme solution. This is generally
followed by a chromatographic step. The introduction
of affinity chromatography as a final and polishing
step has become de rigour in the last 2-3 decades
48,49
.
This kind of protocol is still widely used and serves
the purpose, if the enzyme is required in milligram
amounts for research needs. For any large-scale appli-
cation, it is necessary to follow somewhat different
sequence
48,49
. One important paradigm shift has been
to use affinity interactions in free solution
50-52
. Such
an approach has been tried for lipases as well
53
. Using
alginate as a smart macroaffinity ligand, it has been
possible to selectively precipitate the lipase from
Chromobacterium and porcine pancreas. The simple,
scalable and one-step purification gave 1.7 and 5.6-
fold purification with 87 and 75% of the total activity
recovered
53
. The other important factors like short
process time, low cost of the macroaffinity ligand
(alginate is cheap and can be recovered for reuse) and
scalability makes this a valuable approach for obtain-
ing adequately purified lipases for production of bio-
diesel.
Another possible approach, which may be worth
trying is the use of expanded bed chromatogra-
phy
48,49,54-56
. The use of stable fluidized beds in this
technique obviates the need for any pre-clarification
or concentration step. If the chromatographic media
uses an affinity ligand, affinity capture of lipases
would be expected to give adequately pure enzyme.
The use of these robust, economical and efficient pro-
cesses and some others of similar nature has been re-
viewed earlier
54-56
. The adoption of these techniques
is considered to be useful in cutting down the cost of
downstream processing (and ultimately production
costs) of enzymes.

Immobilized enzymes
42,51,57

Linking enzymes to insoluble matrices by a variety
of methods (such as adsorption, covalent coupling and
entrapment) provides several advantages. In numer-
ous cases, the enzyme becomes more stable. In the
context of transesterification, when the media is pre-
dominantly non-aqueous, stability is a major issue. In
aqueous media, where enzymes are freely soluble,
immobilization provides with a means to recover the
biocatalyst by centrifugation or filtration. In anhy-
drous media, enzyme molecules, though mostly in-
soluble, tend to clump together. Thus, immobilization
is a good way of increasing the surface area of the
biocatalyst. Bosley and Peilow
58
have provided a
critical review and necessary protocol for immobiliza-
tion of lipases. Their recommendation includes the
use of celite or Accurel
TM
(macroporous polypropyl-
ene). A protocol for lipase immobilization by sol-gel
technique is also described. It is also possible to ex-
tensively cross-link the enzyme to obtain insoluble
chemical aggregates, which often show the advantage
of enhanced stability and reusability without increas-
ing the required reaction volume (as these do not have
any volume occupying carrier). A recent innovation in
this area is CLEC
TM
(cross-linked enzyme crystal)
developed by Altus biologicals (USA)
59
. CLEC
TM

form of lipases from Pseudomonas cepacia (CLEC-
PC) and Candida rugosa (CLEC-CR) are also avail-
able
60,61
.

Effect of presence of solvent on biodiesel
production
Biodiesel production using enzymes from oil and
alcohol has been tried in the presence and absence of
solvent. Methanolysis of tallow oil, using Mucor me-
hei lipase in hexane has led to 77.8% ester yield
19
.
Lately, solvent-free transesterification reaction is fa-
voured by many workers, since it is more economical.
Also, the toxicity and flammability of organic sol-
vents, and product recovery without further organic
solvent evaporation favours the use of solvent-free
reaction
62
. znur et al.
18
reported alcoholysis of cot-
ton seed oil in a solvent-free medium, using immobi-
lized Candida antarctica lipase with 92% of the total
ester yield. The use of Rhizopus oryzae lipase (whole
cell biocatalyst) for the synthesis of methyl esters
(MEs) in a solvent-free system led to 71% MEs after
165 hr reaction at 37C with stepwise addition of
methanol
63
. Table 3 lists and compares the ester yields
obtained by alcoholysis with various lipases in the
presence and absence of solvent. For alkali-catalyzed
transesterification, the glycerides and alcohol must be
substantially anhydrous because water produces soap.
The soap reduces the catalytic efficiency by consum-
ing the catalyst and, increases viscosity, gelation,
SHAH et al.: ENZYMATIC TRANSESTERIFICATION FOR BIODIESEL PRODUCTION


397
thereby causing difficulty in glycerol separation. Nel-
son et al.
19
have reported the solvent-free lipase-
catalyzed methanolysis of oils and fats to be sensitive
to water. In contrast, Hsu et al.
64
reported water activ-
ity (a
w
) to be an important parameter for methyl ester
synthesis. Water activity <0.5 led to 98% conversion
to methyl esters. The essentiality of presence of water
has also been demonstrated, while using Rhizopus
oryzae
62
, Candida rugosa
62
, Psuedomonas fluores-
cens
17, 62
and P. cepacia
62
for transesterification.

Some biochemical challenges
While lipases catalysed transesterification reactions
have been extensively used for production of drug
intermediates, biosurfactants and designer fats, the
alcoholysis of the fat/oil has several complex factors,
which need to be taken into account for a viable proc-
ess development:

Inhibition by alcohols
Short chain alcohols like methanol and ethanol are
known to inactivate enzymes. Shimada et al.
25
and
Belafi-Bako et al.
65
have suggested stepwise or con-
tinuous addition of methanol. In the case of latter
work, the highest conversion (97%) was obtained by
continuous addition strategy as compared to the pro-
tocol when necessary methanol was added in eight
steps (94% conversion).
Table 3Effect of presence and absence of solvent on enzyme-based biodiesel production

Alcohol Oil source Solvent Lipase Yield References

Methanol Tallow Hexane Mucor miehei 94.8 16
Tallow None Mucor miehei 19.4 16
Cotton seed None C. antarctica 92 15
Restaurant grease None Thermomyces lanuginosa 4 20
(immobilized)
Restaurant grease None C. antarctica (immobilized) 27 20

Ethanol Tallow Hexane Mucor miehei 98 16
Tallow None Mucor miehei 65.5 16
Restaurant grease None Thermomyces lanuginosa 87 20
(immobilized)
Restaurant grease None C. antarctica (immobilized) 76 20

Propanol Restaurant grease None Thermomyces lanuginosa 87 20
(immobilized)
Restaurant grease None C. antarctica (immobilized) 79 20

Isopropanol Tallow Hexane C. antarctica 51.7 16
Tallow None Mucor miehei 90.3 16
Restaurant grease None Thermomyces lanuginosa 61 20
(immobilized)
Restaurant grease None C. antarctica (immobilized) 59 20

Butanol Restaurant grease None Thermomyces lanuginosa 90 20
(immobilized)
Restaurant grease None C. antarctica (immobilized) 56 20

2-Butanol Tallow Hexane Mucor miehei 83.8 16
Tallow None Mucor miehei 96.4 16
Restaurant grease None Thermomyces lanuginosa 97 20
(immobilized)
Restaurant grease None C. antarctica (immobilized) 89 20

INDIAN J. BIOCHEM. BIOPHYS., VOL. 40, DECEMBER 2003


398
Inhibition by glycerol (and its recovery)
Belafi-Bako et al.
65
reported that while alkyl esters
did not inhibit the biotransformation, the second
product glycerol did inhibit both rate and extent of
conversion. Their solution was use of a membrane
reactor to continuously remove glycerol in order to
relieve product inhibition. It was found that 85 mL L
-1
flow rate at 50C with a flat sheet membrane gave
good results and 100% glycerol recovery rate was
achieved after about 150 min.

Conclusion
The production of biodiesel starting with (i) a local
or regional source of oil/fat, which is either non-
edible or where availability exceeds its consumption;
and (ii) waste or spent cooking oil seem an attractive
approach for obtaining green fuel. As in many bio-
technological processes, the key is to develop self-
reliance in production of the biocatalyst. Unfortu-
nately, as often happens, lopsided emphasis on more
fashionable areas in biology has resulted in neglect of
the art and science of obtaining enzymes.

Acknowledgement
The financial support provided by Council of Sci-
entific and Industrial Research (CSIR) to Shweta
Sharma and Shweta Shah in the form of Senior Re-
search Fellowship is duly acknowledged. Ms. Arti
Sharma is thanked for her help in figure illustration.
The funds provided by Council of Scientific and In-
dustrial Research (Extramural and Technology Mis-
sion on Oilseeds, Pulses and Maize), Department of
Science and Technology (DST) and Department of
Biotechnology, Government of India organizations
are gratefully acknowledged.

References
1 http://www.biodiesel.org/resources/faqs/default.shtm
2 http://www.nationalcleancities.org/govtrelat/biodiesel.asp
3 Standard Specification for Diesel Fuel Oils (2002) in Annual
Book of ASTM Standards, Vol. 5.2. (www.astm.org)
4 Bureau of Indian Standards (BIS) Catalogue (2002), Indian
Standard Diesel Fuel Specification (Fourth revision), Manak
Bhawan, 9 Bhadur Shah Zafar Marg, New Delhi 110 012,
India
5 Srivastava A & Prasad R (2000) Renew Sustain Ener Rev 4,
111-133
6 Pryde E H (1983) J Am Oil Chem Soc 60, 1557-1558
7 Schwab A W, Dykstra G J, Selke E, Sorenson S C & Pryde E
H (1988) J Am Oil Chem Soc 65, 1781-1786
8 Crabbe E, Nolasco-Hipolito C, Kobayashi G, Sonomoto K &
Ishizaki A (2001) Process Biochem 37, 65-71
9 Gubitz G M, Mittelbach M & Trabi M (1998) Bioresour
Technol 1998, 67, 73-82
10 Openshaw K (2000) Biomass Bioener, 19, 1-15
11 Giannelos P N, Zannikos F, Stournas S, Lois E & Anas-
topoulos G (2002) Crops Prod 16, 1-9
12 Linko Y Y, Lms M, Wu X, Vosukainen W, Sappl J &
Linko P (1998) J Biotechnol 66, 41-50
13 Abigor R P, Vadia P, Foglia T, Hass M, Jones K, Okefa E,
Obibuzor J & Bator M (2000) Biochem Soc Trans 28, 979-
981
14 Samukawa T, Kaieda M, Matsumoto T, Ban K, Kondo A,
Shimada Y, Noda H & Fukuda H (2000) J Biosci Bioeng 90,
180-183
15 Antolin G, Tinaut F V, Briceno Y, Castano V, Perez C &
Ramirez A I (2002) Bioresour Technol 83, 111-114
16 Mittelbach M (1990) J Am Oil Chem Soc 67, 168-170
17 Iso M, Chen B, Eguchi M, Kudo T & Shrestha S (2001)
J Mol Catal 16, 53-58
18 znur K, Tter M & Aksoy H A (2002) Bioresour Technol
83, 125-129
19 Nelson L A, Foglia A & Marman W N (1996) J Am Oil
Chem Soc 73, 1191-1195
20 De B K, Bhattacharya P K & Bandhu C (1999) J Am Oil
Chem Soc 76, 451-453
21 Foidl N, Foidl G, Sanchez M, Mittelbach M & Hackel S
(1996) Bioresour Technol 58, 77-82
22 Wu W H, Foglia T A, Marmer W N, Dunn R O, Goering C E
& Briggs T J (1998) Crops Prod 75, 1173-1178
23 Widyan M I & Al-Shyoukh A O (2002) Bioresour Technol
85, 253-256
24 Wu W H, Foglia T A, Marmer W N & Phillips J G (1999) J
Am Oil Chem Soc, 76, 517-521
25 Shimada Y, Watanabe Y, Sugihara A & Tominaga Y (2002)
J Mol Catal B Enz 17, 133-142
26 Hui Y H (1996) in Baileys Industrial Oil and Fat Products
4, 78-394
27 Rosenthal A, Pyle D L & Niranjan K (1996) Enzyme Microb
Technol 19, 400-420
28 Sharma A, Khare S K & Gupta M N (2001) Bioresour Tech-
nol 78, 1-3
29 Sharma A, Khare S K & Gupta M N (2002) J Am Oil Chem
Soc 79, 1-4
30 Sharma A, Khare S K & Gupta M N (2002) Bioresour Tech-
nol 85, 27-329
31 Lusas E W, Watkins L R, Koseglu S S, Rhee K C, Hernan-
dez Riaz M N, Johnson T & Doty S C (1997) INFORM, 8,
280-305
32 http://www.hempcar.org/biofacts.shtml
33 http://earthsci.org/energy/biofuels/biofuels.html
34 http://www.biodiesel.com/biodiesel_fuel.htm
35 Aksoy H A, Kahraman I, Karaosmanoglu F & Civelekoglu H
(1988) J Am Oil Chem Soc 65, 936-938
36 Fukuda H, Kondo A & Noda H (2001) J Biosci Bioeng 92,
405-416
37 Freedman B, Pryde E H & Mounts T L (1984) J Am Oil
Chem Soc 61, 1638-1642
38 Vulfson E N (1994) in Lipases-their Structure, Biochemistry
& Application (Woolley P & Petersen S B, eds.), 271-288,
Cambridge Univ. Press, Cambridge
39 Klibanov A M (1986) Chemtech, 16, 354-359
40 Gupta M N (1992) Eur J Biochem 203, 25-32
41 Gupta M N & Mattiasson B (1992) in Bioanalytical Applica-
tions of Enzymes (Suelter C H & Kricka L, eds.), Vol. 36, pp
SHAH et al.: ENZYMATIC TRANSESTERIFICATION FOR BIODIESEL PRODUCTION


399
1-34, John Wiley and Sons Inc. New York
42 Cabral J M S & Kennedy J K (1991) in Protein Immobiliza-
tion: Fundamentals and Applications (Taylor R F, ed.), pp
73-105, Marcel Dekker Inc., New York
43 Tyagi R & Gupta M N (1998) Biochemistry (Moscow) 63,
334-344
44 Gupta M N (ed.) (1993) Thermostability of Enzymes.
Springer Verlag: Heidelberg
45 Khmelnitsky Y L, Welch S H, Clark D S & Dordick J S
(1994) J Am Chem Soc 116, 2647-2648
46 Mason T J (1997) Chem Soc Rev 26, 443-451
47 Parker M C, Besson T, Lamare S & Legoy M D (1996) Tet-
rahedron Lett 37, 8383-8386
48 Roy I & Gupta M N (2000) Curr Sci 78, 587-591
49 Roy I & Gupta M N (2003) in Isolation and Purification of
Proteins (Hatti-Kaul R & Mattiasson B, eds.), pp 57-94,
Marcel Dekker, Inc. New York
50 Gupta M N & Mattiasson B (1994) Chem Indus 17, 673-675
51 Gupta M N & Mattiasson B (1994) in Highly Selective Sepa-
rations in Biotechnology (Street G, ed.), pp 7-33, Chapman
and Hall: London
52 Galaev I Y, Gupta M N & Mattiasson B (1996) Chemtech
26, 19-25
53 Sharma S & Gupta M N (2000) Biotechnol Appl Biochem
161-165
54 Chase H A (1994) Trends Biotechnol 12, 296-303
55 Roy I, Sardar M & Gupta M N (2000) Enzyme Microb Tech-
nol, 27, 53-65
56 Roy I & Gupta M N (2002) J Chromatogr A, 950, 131-137
57 Kennedy J F, Melo E H M & Jumel K (1989) in Biotechnol-
ogy and Genetic Engineering Reviews, pp. 297-313, Inter-
cept, New Castle Upon Tyne
58 Bosley J A & Peilow A D (2000) in Methods in Non-aqueous
Enzymology (Gupta M N, ed), pp 52-69, Birkhauser Verlag,
Basel
59 Lee T S, Vaghjiani J D, Lye G J & Turner M K (2000) En-
zyme Microb Technol 26, 582-592
60 St Clair N L & Navia M A (1992) J Am Chem Soc 114,
7314-7316
61 Margolin A L (1996) Trends Biotechnol 14, 223-230
62 Kaieda M, Samukawa T, Matsumoto T, Ban K, Kondo A,
Shimada Y, Noda H, Nomoto F, Ohtsuka K, Izumoto E &
Fukuda H (1999) J Biosci Bioeng 88, 627-631
63 Ban K, Kaieda M, Matsumoto T, Kondo A & Fukuda H
(2001) Biochem Eng J 8, 39-43
64 Hsu A-F, Jones K, Foglia T A & Marmer W N (2002) Bio-
technol Appl Biochem 36, 181-186
65 Belafi-Bako K, Kovacs F, Gubicza L & Hancsok J (2002)
Biocat Biotrans 20, 437-439